The use of smartphone for the detection of biological constituents is becoming a useful tool as a point-of-care (POC) device and diagnostics. When combined with microfluidic paper analytic devices (μPAD) and particle immunoassays, we have the ability to detect bacterial pathogens with sensitivity and specificity. Environmental conditions as well as variability in smartphone imaging and the cellulose in paper microfluidics however can sometimes easily interfere with the detection of small signal changes. Combining this issue with the detection of pathogens in blood (our model biological sample of interest) becomes difficult with such a platform because of the complexity of the sample matrix. However, in this research we take a novel approach at utilizing polystyrene’s auto-fluorescence and the high energy of UV LEDs in a particle immunoassay in order to increase our signal change. We first characterized how the smartphone actually responds to UV light (275-385 nm) with respect to the RGB components in its images. We were then able to determine a favorable response using the 385 nm UV LED. The detection of green fluorescence by polystyrene particles was possible by analyzing the smartphone’s image in the green channel. There was a significant difference in signal change with blood samples including polystyrene versus just blood samples with a normalized signal intensity change of 2.5 (150%). The detection of polystyrene fluorescence was translated into a field deployable prototype, where preliminary trials showed promising results in detecting Escherichia coli in blood with a current limit of detection of 50 CFU/ml. With further experimentation and optimization the limit of detection could be improved to 10 CFU/mL, making it a very useful tool in the detection of blood borne pathogens to prevent complications with onset bacteremia and the more serious cases of sepsis. This assay platform could provide an easy to use solution with detection in a short time (assay time of 1 min) compared to the lengthy blood culture monitoring or biomarker detection.

The purpose of this thesis project is to demonstrate the ability to utilize electrophoresis in a cellulose paper microfluidic chip to manipulate charged particles. Materials were selected and a manufacturing protocol was created to successfully apply the electric field onto the paper chip. Experiments were performed to characterize the separation rates for charged, colorimetric dye, Orange G in the membrane as a function of an applied electric field, dye concentration, and distance traveled. The experiments confirmed that the electric field can be applied to the chip and particle separation rates were characterized. Next, the determined rates results were used to design a device that used a transverse electric field to the flow direction to separate Orange G into a collection channel. Results showed that electrophoresis can be used to separate the flow of charged particles on a paper microfluidic device. In conclusion, the application of electrophoresis was shown to be successful. An approach to be utilized as a sample treatment to improve the detecting capability of low cost paper devices for a more accurate diagnostic test in the developing world.

The field of microfluidics is expanding into what is known as paper microfluidics. This uses a paper platform rather than materials (i.e. PDMS, PMMA) that are commonly used in microfluidics research. Current devices require an expensive manufacturing process and external sources to power the device. Such devices are not practical in low resource environments. As a consequence, it is the goal of this Thesis to develop a three-dimensional, multiplexed assay chip using nitrocellulose membranes. This device comprises of multiple layers of nitrocellulose membranes with defined fluidic channels. The multiple layers are bound together using double backed tape, and imbedded between the layers are conjugate reagents. In the detection region both antibodies and aptamers were evaluated. The fiberglass pad where conjugate reagents would be contained, were initially saturated in dye. As sample was inputted into the three-dimensional chip, the fluid path could be visualized. Without the use of the conjugate pad the chip’s four detection regions showed detection within one minute of one another. However, the addition of this fibrous pad skewed time points dramatically. The hypothesis that a three-dimensional chip could be designed to detect different biomarkers in a multi-analyte sample was satisfied. However, simultaneous detection was only possible if the conjugate pad was either neglected or, possibly, a different material was used. Additionally, current lateral flow assay technologies, another research area that paper microfluidics spawns from, use antibodies in order to capture biomarkers in sample and provide visual signal to the user. However, antibodies are sensitive to denaturation with pH and temperature, whereas aptamers can withstand much more extreme environmental conditions. A two-dimensional nitrocellulose chip was designed to compare antibodies and aptamers as capture reagents to detect VEGF, using colloidal gold as a particle to visualize detection. Both monoclonal and polyclonal anti-VEGF antibodies were used and showed no signal. On the other hand, the anti-VEGF aptamer produced a visual signal when conjugated to biotin on its 5’ end. This data was further validated by a separate project analyzing the binding kinetics of the antibody and the aptamer using Surface Plasmon Resonance. Therefore, the hypothesis that aptamers could be used as a possible capture reagent in a paper microfluidic chip for the detection of VEGF was satisfied.

In the United States and other developed countries, despite great efforts in time and funding for the prevention of foodborne and airborne diseases, there is still an unacceptable level of common pathogens spread via food, water, and air. To this end, lab-on-a-chip (LOC) technologies were developed for field-deployable assays and point of care diagnostics. These devices have potential applications in hospitals, agricultural farms, processing plants, and even on fields of battle. Two successful types of assays in the recent years towards point of care diagnostics are immunoassays and nucleic acid detection assays. In the Appendix A, we demonstrated a complete, field-deployable particle immunoassay encased within a microfluidic chip that detects small quantities of Salmonella Typhimurium in poultry fluid samples. Because the necessary reagents are pre-loaded and the test and negative control channels are fed by a single sample inlet, single pipetting of sample is possible. This assay demonstrated a 10 CFU/mL limit of detection, which is considerably lower than PCR and enzyme-linked immunosorbent assay (ELISA). Total assay time, including sample reading in an integrated handheld device, was 10 minutes, which was much lower than conventional methods. Because of the simplified protocol and assay time, this biosensor has potential in clinical and field diagnostic applications. In Appendix B, we fit the particle immunoassay to test for Influenza A H1N1/2009 virus and included aerosol sampling from a scaled-down mock classroom. To make the assay field deployable, we used an iPhone for signal detection. The detection limit of the assay was 1 pg/mL (10 pg/mL using the iPhone), which is well below the limit of detection for RT-PCR. This protocol demonstrated that immunoassays can be effective in the presence of interfering dust particles and that viruses can be collected from aerosol with minimal sample preparation. In Appendix C, we demonstrated that paper microfluidics, a newer vision of microfluidics, is a cheap and easy method to extract nucleic acid from S. Typhimurium in a variety of samples, including poultry packaging liquid, whole blood, and feces. Fluorescent detection with an iPhone allows for field and clinical testing. This protocol interfaces with rapid PCR and is a true diagnostic tool.

Les propriétés du papier - pompe capillaire, prix et disponibilité - lui permettent de répondre à tout type de contraintes logistiques et économiques d'un diagnostic médical de terrain. En amont de l'application, nous avons étudié les écoulements dans des géométries confinées par des barrières de cire. Une focalisation hydrodynamique associée à l'évaporation, crée un effet de concentrateur atteignant un facteur d'amplification de 1000. La continuité du diagnostic depuis le terrain jusqu'au laboratoire est assurée par des dispositifs hybrides papier-microsystèmes. Dans cette thèse, l'élution d'échantillons séchés dans le papier est quantifiée et sa compatibilité avec différentes fonctions microfluidiques démontrée. Nous nous sommes intéressés à deux applications de diagnostic : la détection d'un biomarqueur cardiaque par immunoessai et celle du virus Ebola par amplification d'acides nucléiques (RT-RPA). Avec un dispositif papier simple et une révélation colorimétrique, la troponine a été détectée jusqu'à une concentration de 1 ng/mL. Le diagnostic précoce de maladies infectieuses est rendu possible par la biologie moléculaire sur papier. Après des développements en laboratoires sur ARN synthétiques, des expériences réalisées en Guinée, sur des échantillons cliniques, à partir de papiers prêts à l'emploi, avec une instrumentation transportable ont atteint une sensibilité à 85.3%. Le multiplexage du diagnostic est obtenu dans des géométries multicouches par la réalisation simultanée de tests et contrôles. Enfin, l'application à d'autres pathogènes comme HIV et la Dengue, a montré les limites du papier et de son environnement biochimique non contrôlé. / Paper properties – such as capillary pump, affordability and availability – made it suitable for medical diagnostics in logistic and economic field constraints. Upstream of application, we studied liquid flows in wax-confined geometries. Hydrodynamic focus coupled to evaporation creates a concentrator effect reaching an amplification factor of 1000. Diagnosis continuity from the point of care to testing laboratory is insured thanks to hybrid paper-microsystem devices. In this thesis, the elution of dried samples in paper is quantified and its compatibility with various microfluidic functions demonstrated. We were interested in two diagnostics application: detection of a cardiac biomarker by immunoassay and of Ebola virus by nucleic acids amplification (RT-RPA). With simple paper devices and a colorimetric signal, Troponin has been detected until a concentration of 1 ng/mL. Early diagnostics of infectious diseases is made possible with molecular biology on paper. After laboratory preliminary developments on synthetic RNA strains, experiments performed in Guinea with clinical samples, from ready to use papers, with carry-on equipments achieved a sensitivity of 85.3%. Multiplexed diagnostics is obtained in multilayered geometries enabling simultaneous tests and controls. Finally, application to other pathogens, like HIV and Dengue, showed paper limits from its uncontrolled bio-chemical environment.

In recent years, there has been a dramatic increase in the use of improvised explosive devices (IEDs) due to ease of synthesis and improved controls placed on commercial/military explosives. Commonly used materials for IED preparations include fertilizers and industrial chemicals containing oxidizers such as ClO3-, ClO4-, and NO3-, as well as other less stable compounds, such as peroxides. Due to these materials having a wide range of volatility, polarity, and composition, detection can be challenging, increasing the amount of time before any analytical information on the identity of the explosive can be determined. Therefore, this research project developed two analytical methods to aid in the rapid detection of multiple explosive compounds. The use of microfluidic paper-based analytical devices (µPADs) allows for the development of inexpensive paper devices utilizing colorimetric reactions, which can perform five or more simultaneous analyses in approximately five minutes. Two devices were developed: one for the detection of inorganic explosives including ClO3-, ClO4-, NH4+, NO3-, and NO2-, and the second device detects high/organic explosives including RDX, TNT, urea nitrate, and peroxides. Limits of detection ranged from 0.4 µg – 20 µg of explosive residue with an analysis time of less than five minutes. Development of a confirmatory method utilizing infusion electrochemical detection-electrospray ionization-time-of-flight mass spectrometry (EC-ESI-TOF MS) and 18-crown-6 ethers to produce guest/host complexes with inorganic ions has also been completed. Utilizing this method the inorganic ions present in many IEDs can be successfully detected as ion pairs, while still allowing for the detection of other high explosives1. Placing an electrochemical detector before the mass spectrometer permits the detection of hydrogen peroxide, an analyte normally difficult to detect through mass spectrometry. Limits of detection ranged from 0.06 ppm - 2 ppm with an analysis time of less than two minutes. The development of these presumptive and confirmatory analytical methods permits the detection of a wide range of components present in IEDs. These methods decrease the amount of time required to relay information on the type of explosives present by simplifying the analysis process in the field and in a laboratory.

No description available.

The purpose of this thesis project was to demonstrate the ability to utilize the capabilities of aptamers so that they may act as capture reagents for paper microfluidic devices. Several characterization experiments were conducted as a precursor before the final experimentation was performed. Paper characterization, manufacturing protocols for printing and heating, as well as 3D chip fabrication were all performed and analyzed. The results confirmed that the control of fluid through a 3D microfluidic device based in nitrocellulose is possible. For the biochemistry portion of this thesis report, antibodies and aptamers were chosen to react with IgE, an antibody that is present in high concentrations in the urine of patients diagnosed with respiratory distress. Antibody chips were successfully created as a baseline lateral flow assay for comparison to new aptamer detector reagents. The aptamer experiments were able to demonstrate that it is possible to utilize the capabilities of aptamers so that they may behave as capture reagents in paper microfluidic devices. Overall, the experiments performed were extremely supportive of the ability to develop the field of paper microfluidics with the use of aptamers so that patient populations across the globe can be more accurately and effectively diagnosed.

3-D paper-based microfluidic devices (micoPADs) are small and portable devices made out of paper that offer a promising platform for diagnostic applications outside of a laboratory. These devices are easy to use, low cost, require no power source, and capable of detecting multiple targets simultaneously. The work in this thesis demonstrated the ability of a 3-D paper-microfluidic platform to simultaneously detect 5 targets. Rubber cord stock was used in conjunction with an acrylic housing unit to apply pressure along the edge of the channel. The indirect pressure application was successful in promoting vertical fluid flow between layers. Average channel development times were recorded between 110 seconds and 150 seconds. The implementation of the 3-D paper-microfluidic platform as a diagnostic device was validated with a colorimetric glucose assay. In a novel application, reagents were deposited onto the 3-D platform via a glucose reagent pencil created by Martinez et al. A visual signal was observed for the successful detection of glucose at a concentration of 1.2 mM. These results offer promise for future work in combing new reagent deposition techniques with a multi-layer paper-microfluidic platform. Overall, this research made advancements in the design of a paper-microfluidic platform capable of the simultaneous detection of 5 targets.

abstract: This work describes efforts made toward the development of a compact, quantitative fluorescence-based multiplexed detection platform for point-of-care diagnostics. This includes the development of a microfluidic delivery and actuation system for multistep detection assays. Early detection of infectious diseases requires high sensitivity dependent on the precise actuation of fluids. Methods of fluid actuation were explored to allow delayed delivery of fluidic reagents in multistep detection lateral flow assays (LFAs). Certain hydrophobic materials such as wax were successfully implemented in the LFA with the use of precision dispensed valves. Sublimating materials such as naphthalene were also characterized along with the implementation of a heating system for precision printing of the valves. Various techniques of blood fractionation were also investigated and this work demonstrates successful blood fractionation in an LFA. The fluid flow of reagents was also characterized and validated with the use of mathematical models and multiphysics modeling software. Lastly intuitive, user-friendly mobile and desktop applications were developed to interface the underlying Arduino software. The work advances the development of a system which successfully integrates all components of fluid separation and delivery along with highly sensitive detection and a user-friendly interface; the system will ultimately provide clinically significant diagnostics in a of point-of-care device. / Dissertation/Thesis / Masters Thesis Biomedical Engineering 2018